BIODIVERSITY GENOMICS EUROPE WP4
Pollinator Communities - Malaise trap sampling
SOP
First version: 5 March 2024 based on HMS-Arthropods SOP 2023
Last version: 09 November 2024
Laura Nájera-Cortazar [BIOPOLIS-CIBIO]
Sónia Ferreira [BIOPOLIS-CIBIO]
Pedro Beja [BIOPOLIS-CIBIO]
Associação Biopolis - CIBIO – Centro de Investigação em Biodiversidade e Recursos
Genéticos [BIOPOLIS - CIBIO]
BIODIVERSITY GENOMICS EUROPE
receives funding from the European Union's Horizon Europe Research and Innovation Action.
https://biodiversitygenomics.eu/
2 | 4.4 Pollinator Communities Sampling
protocol / PlutoF Go
Table of contents
Introduction 2
Sampling Design 4
Permits and documentation 6
Before starting 7
Setting Malaise traps and sample collection 9
Shipment 12
Registering samples in PlutoF Go app 13
Acknowledgements 16
References 16
Useful contact 16
Introduction
The Biodiversity Genomics Europe (BGE) Consortium has the aim of accelerating the use of
genomic science to strengthen understanding of biodiversity, monitor its status, and develop
informed strategies to address its decline. One of the main objectives is establishing
international biodiversity genomics networks, data generation and pipelines to characterize
biodiversity, improving management of interventions and biomonitoring programs by the
application of genomic tools. The present standard operating procedure (SOP) is adapted from
the High Mountain Systems - Arthropod sampling with Malaise traps SOP (Najera-Cortazar et
al. 2024), developed within the BGE.
Pollinators are crucial for terrestrial ecosystems. Both ecologically and agriculturally, they
perform important functions that involve many classes of interactions, and support the majority
of the global plant diversity (Ollerton 2017). Insects are one of the most important groups of
pollinators (Ollerton 2017). It has been evaluated that insect biodiversity changes are mostly
driven by climate change and intensive human land-use, including habitat loss by land
transformation and agricultural intensification (Goulson et al., 2015; Ollerton 2017; Outhwaite et
al., 2022). Given the intensification and homogenisation of agricultural land uses in the
countryside, the use of gardens as refugia for biodiversity in urban areas might become of
particular importance. In fact, given the increasing intensity of farming across Europe, involving
for instance increases in field size, loss of non-agricultural habitats (e.g., hedges, woodlots), and
increasing use of agro-chemicals, among other changes, we expect that urban habitats will be
increasingly important for a range of species that are now rare or absent on farmland.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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The Pollinator Communities case study will analyze pollinator diversity using DNA barcoding
(Herbert et al., 2003) and metabarcoding (Taberlet et al., 2012) techniques, using bulk samples
of specimens collected during fieldwork sampling (Young and Herbert, 2022). Specifically, the
study will contribute to: i) Enhance the inventory of pollinators (and other arthropods) using
European urban and agricultural habitats, including exotic species; ii) set a baseline for future
monitoring efforts on pollinators (and other arthropod) temporal trends in European urban and
agricultural habitats; iii) to quantify differences in pollinator community attributes between urban
and agricultural habitats (e.g. Alpha and beta diversities); iv) to identify pollinator species traits
associated with urban living; and v) to engage citizen scientists and the wider community in
pollinator sampling activities.
Figure 1. Examples of Malaise traps set in an Urban garden (left) and in an Agricultural field (right)
in Seia, Portugal.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Sampling Design
The Pollinator Communities case study will involve flying arthropod pollinators sampling with
Malaise traps (Figure 2) set in pairs of sites across Europe. A Malaise trap (Figure 2) is a
tent-like trap that will direct arthropods to go to the top of the trap, for them to fall into a
collection tube attached to the top central section of the trap, containing 96% ethanol or higher
concentration (Najera-Cortazar et al., 2024). For the BGE, we are following the procedures
stated in the Global Malaise Trap program where you can find further information about the
program. The design for Malaise trap samping was first described in the High Mountains
Systems - Arthropods SOP (Najera-Cortazar et al., 2024). Detailed information about how to set
the Malaise traps is described in page 9.
Figure 2. Scheme of a Townes Style Malaise trap and its dimensions (left); and example of a
Malaise trap set in an agricultural field in Portugal (right).
Each collaborator is expected to select and sample at least three different pairs of sites for five
weeks each. Each pair of traps should correspond to one site in an Urban Garden and another
one in an Agricultural Field (Figure 1 and 3), which should be located at no more than 20 km
from each other. If there is only one pair of Malaise traps available, sampling should be done in
a rotating manner: One site pair sampling for five weeks, then moved to a different site for five
weeks, and then to another one for another five weeks. In total, it would be 15 weeks of
sampling. Each trap will collect flying arthropods through a 500 mL collection tube filled (around
300 mL) with at least 96% ethanol (i.e. 96% or more), that will be used for characterizing the
pollinator community using DNA metabarcoding. Ethanol volume can be adjusted in the
following week of sampling if it was too much or too little for that site’s trap.
Samples will be collected each week, by swapping the “filled” tube of each Malaise trap for a
new one. Sample tubes must be collected during five weeks on the same day of the week
during all the sampling time (e.g. tube collection from one or all pairs will be each Monday).
Each pair set will generate two tubes/samples per week, giving a total of 10 bulk samples
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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per five weeks of sampling, that will be sent for DNA metabarcoding analyses. Please note:
once removed from the Malaise trap, tubes should not be reopened for any reason to
avoid contamination. Make sure that the arthropod mass is submerged in ≥96% ethanol and
store the samples safely at room temperature in a fresh place, away from light exposure.
The definition of Urban Garden and Agricultural Field sites are dependent on the types of
environments available in each particular area sampled. The following conditions should apply
for site selection, except if otherwise agreed with the project’s coordinators, Prof. Pedro Beja
● Agricultural Field sites should be located in areas where land cover is predominantly
agricultural (e.g., annual crops, permanent crops) within about 2 km of the site, and to be
in or at the edge of an agricultural field where productive agricultural areas (i.e.,
excluding farmhouses, hedges, woodlots, ponds, and other non-productive habitats) are
dominant within about 100 m of the site. Any type of agricultural site can be considered
(e.g., cereals, vegetables, fruit orchards, among others), but excluding areas dominated
by greenhouses, livestock pastures, and urban vegetable gardens.
● Urban Garden sites should be located in areas where land cover is predominantly urban
(e.g., housing, roads, urban green areas) within about 2 km of the site, and to be in a
garden where green areas are dominant within about 100 m of the site. Gardens can be
botanic gardens, public leisure gardens, or private backyard gardens, among others.
Ideally, the garden should have a mixture of habitats, including woodlots or at least
isolated trees, flower beds and meadows.
Figure 3. Sampling design, showing an example of an Agricultural field (left) and an Urban garden
(right), separated from up to 20 km. The blue circle denotes land cover dominance.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Materials
List of materials needed for Malaise trap setting and sample collection
A. A pair of Malaise traps (ez-Malaise Trap II mode, see BugDorm store)
B. Extra cords, metal stacks, etc. for mounting (more to read in the section “Before sampling”)
C. 500 mL sterile collection tubes (see Wide-Mouth LDPE Bottles with Closure link)
D. ≥ 96% Ethanol
E. Sticky labels with predefined QR codes to be read by the PlutoF Go app
1
(.pdf will be provided by
BIOPOLIS-CIBIO)
F. Transparent tape (to provide extra fixation for the sticky labels on the tube)
G. Cable ties
H. Hammer
I. Gray duct tape
J. Aluminum foil (Optional, to cover the tubes during sampling)
The PlutoF platform (Kessy et al. 2010) will be used as a workbench for processing the
metadata. It includes a mobile app, PlutoF Go, that will be used for data entry during fieldwork.
QR codes will be supplied by email to each partner institution for adding them to each collection
tube, prior to sampling. More information regarding PlutoF usage, labels and procedures will be
provided further in the document.
Permits and documentation
It is highly important to make sure all the permits and necessary documentation are ready
before sampling. To prevent any delays, check regulation and start processing your permits as
soon as possible.
Permission from local authorities, property owners, rangers, or protected areas stakeholders
can be another factor of potential delay or cancellation. Make sure you formalize the
authorization to sample on your selected area on time, and if possible, obtain a written
confirmation.
Have copies of any legal documentation ready in case they are needed.
1
The PlutoF Go app can be found for Android, and for iOS systems. Instructions and other information
can be found in the link provided. A quick guide for PlutoF Go is provided in this document.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Before starting
The following instructions are taken from Najera-Cortazar et al. (2024). Make sure you have all
sampling materials organized, to have sorted fieldwork logistics (e.g., vehicle, budget,
personnel, a fixed day of the week for collection, etc.), to consider habitat characteristics,
sampling location, storing equipment, obtaining all necessary permissions/authorization for
collecting specimens (see previous section).
It is advisable to have several options of pair sites chosen when possible, as backups. This is
particularly relevant if the sites selected for this project are new for the team, as is important to
consider anthropogenic factors that may disrupt the traps, like hiking visitors, private
landowners, cattle, vandalism, etc. It is important to ensure that each pair site is accessible
during its five weeks of sampling, and that tube collectors will be available during that period.
Malaise traps ordered for the BGE project contain a plastic grid (located in the collection
mechanism in the top of the trap) that comes within the trap. For the Pollinator communities
sampling it needs to be removed prior to setting the trap. Otherwise arthropods pollinators larger
than the size of the grid will not be sampled. In Figure 4 is shown an example of how to remove
the plastic grid and to secure the mesh back using cable ties.
Figure 4. Example of how to remove the plastic grid included in the Malaise trap.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
8 | 4.4 Pollinator Communities Sampling
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To optimize time of sampling and storing, the QR codes stickers should be placed on the sterile
tubes and reinforced with tape prior to sampling. Partner’s coordinators must be registered in
the PlutoF platform (“Become a user”: Register → fill in details) in order to have access to the
corresponding project (Pollinator Communities - “name of partner institution”) when using the
PlutoF Go app, to appear as a collector. Alternatively, project coordinators can add “persons”
into the platform (Menu “Persons” → Add → fill in details) if the collector will be a person that will
be only involved in fieldwork. Make sure you have downloaded the app and enter your personal
data correctly. Further information on PlutoF Go is given on page 13. Support will be provided
whenever needed before, during and after the fieldwork (see contact details at the
end of this document).
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Setting Malaise traps and sample collection
The design of the trap relies on insects being attracted to the highest and brightest part of the
trap. When setting up the trap, ensure that the part that collects the pollinators (the trap head) is
facing uphill. Ideally, position the trap perpendicular to the path of arthropod’s flight, in areas
with minimal undergrowth, such as forest edges, clearings, or elevated locations. Take into
account potential disruptions by wildlife, humans, as well as the direction of the prevailing winds.
Once you have chosen a location, follow the Malaise trap instruction sheet to securely assemble
the trap. A video of how to set up a Malaise trap can be found in this link. When possible, fasten
the front and/or back ropes to nearby trees to provide extra support, and use metal pegs to
attach the bottom rings of the traps to the ground (Figure 5, left), placed in opposite directions to
the trap. Particularly if your sites are located in areas of strong winds, it is advisable to attach
the trap poles to a 1 m - 1.5m stake or post at the highest points to prevent the trap from
toppling over. Use gray tape to reinforce the joints of the trap's metal frame and increase its
stability and resilience (Figure 5, right).
Figure 5. Metal stack/peg placed at one of the extremes of the Malaise trap (left). Gray tape placed
in the joints of the trap metal structure for reinforcement (right).
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
10 | 4.4 Pollinator Communities Sampling
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When assembling the trap, make sure to put tension in all the extremes, and that the head
structure in the front (i.e. the tent-like metal structure) is parallel to the tail structure support. To
ensure the entrance is fully open, imagine you are an insect and fly into the trap towards the
trap head (Figure 6), making sure that there is a clear path/entry to the collection tube at the top
of the trap). Check to not over-stretch the mesh, as this will most likely block the path too.
Figure 6. Arthropod view to “the light” path.
Once the trap is set, carefully attach the BGE.PC labeled collection tube tightly to the trap head,
filled with ≥96% ethanol, and secure it with the white straps on the trap (Figure 7). Take pictures
of the trap set and its location. Remember to begin collecting on a day of the week when you
can reliably return for the duration of the sampling period (five weeks). Additionally, you can
cover the tube with aluminum foil, to keep a more stable temperature.
Ethanol recommendations: as previously mentioned, the tubes should be filed approximately
with 350 mL of ethanol. After the first collection, you would be able to monitor if you needed
more ethanol or less, so you can adjust the quantity of ethanol per tube, and per site. During hot
weeks, ethanol could evaporate faster, or you might have more specimens than expected, so
you can add more or less depending on how your collections are.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Figure 7. Example of a collection tube correctly placed and secured in the trap using the white
straps around it.
Every week, two tubes will be collected per urban/agricultural pair site, making a total of two
tubes per week, during five weeks (10 tubes per pair). Take some extra tubes with you to the
field in case something happens when swapping tubes. Each collection tube should be handled
carefully to prevent contamination. Remember: when collecting the tubes from the traps each
week, tubes should be filled with ethanol, closed and not opened again.
Each pair of sites should be selected under previous agreement with landowners/stakeholders
and having any necessary permit/documentation. Despite this, there could always be problems
or eventualities with the Malaise traps (e.g. vandalized, cattle playing ground, extreme weather).
If something happens to the trap, you can set one of the replacements nearby, and take a note if
the sample was retrieved or lost. Institutional signs may also help to protect the traps.
After sampling, make sure that all the tubes are well closed, have enough ethanol to cover the
specimens (top it up if needed), and store them at room temperature, away from light exposure.
In the PlutoF platform, complete any missing information.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Shipment
For the BGE HMS Arthropods and the Pollinator Communities projects, samples will be shipped
to Dr. Brent Emerson, based in the Institute of Natural Products and Agrobiology (Instituto de
Productos Naturales y Agrobiología, IPNA-CSIC), in Canary Islands:
Brent Emerson
Island Ecology and Evolution Research Group
Instituto de Productos Naturales y Agrobiología (IPNA-CSIC)
C/Astrofísico Francisco Sánchez 3
La Laguna, Tenerife, Canary Islands, 38206, Spain
e-mail: [email protected]
Before shipping the samples, make sure that the IPNA-CSI lab has
confirmed the availability to receive the samples (partners will ship
samples in different rounds). Please confirm this with project
Emerson.
For shipping, It is needed to remove as much ethanol as possible
from each tube, leaving only enough to keep the arthropods
“moist”. To do this, please make sure you are working in a sterile
environment, i.e. laboratory, and only under these conditions and
at a unique time, open each tube using gloves to decant the
supernatant ethanol.
Full instructions of how to ship Malaise traps tubes are described in the “Malaise traps - Bulk
samples Shipping instructions” living document, please refer to the information when needed.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
13 | 4.4 Pollinator Communities Sampling
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Registering samples in PlutoF Go app
PlutoF is an online data management and computing service provider for biological data. PlutoF
Go is the app that will be used to record samples directly in the field. Before using the app, the
data collector should be registered on the PlutoF website. This can be done by the user in the
option “Become a user”, or to be added by the BGE project manager directly on her/his
workbench
2
. You can fill all the available information within the Bulk sample option, but it is
required at least to have the data detailed below:
1. Open the PlutoF Go app
2. Go to Add material sample box (If this option is not visible in the main page, go to
Settings, scroll down to Material sample, activate Enable material sample gathering and
make sure the Bulk form is highlighted in green as well).
3. The Location button will show your position in the map. You
should record all the necessary information at the moment of the
sample collection, therefore it should capture the coordinates
detected by your device’s GPS. If there is no internet signal you
can still get the coordinates by pressing the compass icon (top
right inside the map box).
2
The PlutoF project manager will be the only one authorized to add any person to the working project.
Any team member will be automatically notified by email when added to any project.
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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4. Start date: insert the collection date (when the
tube is removed from the trap).
5. Project: choose the “Pollinator Communities -
Institution” option. Choose the project according to
your institution acronym (compulsory field).
6. Choose form: select “Bulk”.
7. Sample ID: click on the code icon and point
your device camera to the corresponding QR code
provided for the sampling. QR codes are unique and
cannot be added multiple times (compulsory field).
8. Subcode: Add the country prefix of your study, point,
Institution prefix, Agricultural/Garden + trap in a consistent
way for all the sampling, for example for Portugal
PT.CIBIO.A1, PT.CIBIO.G1:
❖ Pair 1: Agricultural Malaise 1 (A1) and Garden Malaise 1 (G1) = these subcodes will correspond to each
BGE.PC00XX sample collected from the same traps, every time you visit them
❖ Pair 2: Agricultural Malaise 2 (A2), and Garden Malaise 2 (G2). And so on until Pair 3… If you sample
three pairs of sites, you will have A1,G1; A2, G2; and A3, G3. Please record location and all the required
details per each trap set
9. Description: Write “Placement date of Malaise trap
[date] or “Placement date of the collection tube [date]”. This
to know how long was the tube sampling in the trap
10. Add Collectors: Any person has to be previously
registered/added in the PlutoF platform
11. Event description: Describe any relevant events
during the sampling week (e.g. strong wind/rain, external
disturbance by human/animal; etc.)
12. Habitat description: Describe any information
about the site you have sampled. For example: type of
garden, % of flower coverage, taxonomic description of plants (if known), type of
agricultural field, types of crops, % of crop coverage, position of trap respective to the
crop land, etc. Pictures of the trap's surroundings (point 14) will help to support this
13. Trap ID: an identifier for each trap, if needed
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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14. Add at least one photo of the Malaise trap and its
environment using the panel that is in the top of the
Material sample menu. Use the camera + icon (top left)
to generate a picture (For example, see Figure 1 and 7).
You can add any extra information or metadata you think
convenient using the media bar (more photos, videos,
etc.), anything you think will be useful for further
assessment of the site, vegetation, etc.
15. Review all the information submitted is accurate
and click “Save” at the bottom of the screen
16. In the main screen, your entries will be waiting in
the queue to be synchronized. Click on the cloud icon on
the top right to do it.
In the image, there is one good sample entry (dark letters,
intense color) that can be synchronized, and another entry
with missing information (red letters, faded color) that will
not be able to sync until the missing information is filled.
This can be done by clicking on the entry and revising the
info submitted.
If you cannot sync a sample, click back to that entry and
check your institutional project is selected. Save, and try to
sync again.
Make sure of having an internet connection to sync your
entries, and try to sync often to ensure the data is saved.
Keep in mind that entries with media files (pictures, videos,
etc.) will take longer to sync, and more internet to load.
17. You are ready for the next site sampling collection!
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
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Acknowledgements
Biodiversity Genomics Europe (Grant no.101059492) is funded by Horizon Europe under the
Biodiversity, Circular Economy and Environment call (REA.B.3); co-funded by the Swiss State
Secretariat for Education, Research and Innovation (SERI) under contract numbers 22.00173
and 24.00054; and by the UK Research and Innovation (UKRI) under the Department for
Business, Energy and Industrial Strategy’s Horizon Europe Guarantee Scheme.
References
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M., Aan, A., Ots, M., Kurina, O., Ostonen, I., Jõgeva, J., Halapuu, S., Põldmaa, K., Toots, M.,
Truu, J., Larsson, K-H., and Kõljalg, U. 2010. PlutoF - a web based workbench for ecological
and taxonomic research, with an online implementation for Fungal ITS sequences. Evolutionary
Bioinformatics, 6, 189 - 196.
Goulson, D., Nicholls, E., Rotheray, E. L., Botías, C. 2015. Qualifying pollinator decline
evidence—response. Science, 348:982. https://doi.org/10.1126/science.348.6238.982
Hebert, P. D., Cywinska, A., Ball, S. L., and deWaard, J. R. 2003. Biological identifications
through DNA barcodes. Proc Biol Sci. 7,270(1512):313-21.
https://doi.org/10.1098/rspb.2002.2218.
Najera-Cortazar, L. A., Ferreira, S., Mata, V., and Beja, P. 2024. Biodiversity Genomics Europe |
High Mountain Systems - Arthropod sampling with Malaise traps. WorkflowHub,
https://doi.org/10.48546/workflowhub.sop.17.2.
Ollerton, J. 2017. Pollinator Diversity: distribution, ecological function, and conservation.
Annu. Rev. Ecol. Evol. Syst. 2017. 48:353–76. https://doi.org/10.1146/annurev-ecolsys-110316-
022919
Outhwaite, C. L., McCann, P., and Newbold, T. (2022). Agriculture and climate change are
reshaping insect biodiversity worldwide. Nature, 605(7908):97-102.
https://doi.org/10.1038/s41586-022-04644-x.
Taberlet, P., Coissac, E., Hajibabaei, M., and Rieseberg, L.H. 2012. Environmental DNA. Mol.
Ecol., 21: 1789-1793. https://doi.org/10.1111/J.1365-294x.2012.05542.X.
Young, M. R. and Hebert, P. D. N. 2022. Unearthing soil arthropod diversity through DNA
metabarcoding. PeerJ. 1,10:e12845. https://doi.org/10.7717/peerj.12845.
Useful contact
Laura Nájera Cortazar | Associação Biopolis - CIBIO – Centro de Investigação em
BIODIVERSITY GENOMICS EUROPE receives funding from the European Union's Horizon Europe
Research and Innovation Action. https://biodiversitygenomics.eu/
